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. 2009 Jul;66(1):73-81.
doi: 10.1016/j.pep.2009.03.001. Epub 2009 Mar 9.

Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1

Samantha J Allen  1 , Sofia Ribeiro, Richard Horuk, Tracy M Handel
Affiliations

Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1

Samantha J Allen et al. Protein Expr Purif. 2009 Jul.

Abstract

Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.

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Figures

Figure 1
Figure 1. Sequence and predicted topology of the chemokine receptor CCR1
Potential N-linked and O-linked glycosylation sites (gray circles) and tyrosine sulfation sites (squares) are highlighted. Chemokines bind to the extracellular surface, whilst G-proteins bind to the cytoplasmic surface, in a region encompassing the second and third cytoplasmic loops, and C-terminal tail of CCR1. Transmembrane helices were positioned according to Vaidehi et al. [21].
Figure 2
Figure 2. Expression of chemokine receptors in different systems
a) CCR1 variants containing different N- and/or C-terminal tags were stably transfected into mammalian cell systems (FlpIn/HEK293, T-Rex/HEK293, FlpIn/T-Rex/HEK293 and pACMV-TetO/HEK293). Where appropriate, cells were induced and cell surface receptor levels detected as described in the experimental procedures. b) Chemokine receptor variants CCR1, CCR2, CCR3 and CCR5 were transfected into HEK293 cells and expression levels of induced and un-induced cells were measured (n=4, shown as mean ± S.D.). Key: CCRXS: CCRX-StrepII, SCCRXH: StrepII-CCRX-His8, HACCRXH: Hemagglutinin-CCRX-His8.
Figure 3
Figure 3. Immunofluorescence staining of CCR1
Cells expressing CCR1 were harvested, permeabilized where indicated, and stained with the following antibodies: anti-CCR1-phycoerythrin antibody (red), nuclear stain (blue) and either the cis Golgi antibody anti-GM130 (green, top panel) or the trans Golgi antibody 58K (green, bottom panel).
Figure 4
Figure 4. Functional studies of CCR1 in HEK293 cells
a) Chemokine binding to CCR1 was studied by the ability of CCL3, CCL7 or CCL7 containing a C-terminal AlexaFluor 647 moiety (CCL7-AF647) to displace radiolabeled CCL3. b) Chemokine-dependent calcium mobilization in CCR1 transfected HEK293 cells. Data were expressed as chemokine concentration versus maximum fluorescence of a calcium-specific dye (n≥3, shown as mean ± S.D.). Except where indicated, CCL14 (9-74) was used.
Figure 5
Figure 5. Purification of CCR1 in different lipid/detergent micelles
a) Coomassie stained SDS-PAGE gel and b) western blot after solubilization and partial purification of CCR1 by Ni-affinity chromatography. The detergents/lipids used for solubilization were as follows: 1) 2% OG, 2) 0.2% DG, 3) 2% Cymal-5, 4) 1% Cymal-7, 5) 1% FC-14, 6) 2% Cyclofos-5, 7) 2% DM, 8) 1% DDM, 9) 1% TDM, 10) 1% DDM + 0.2% CHS, 11) 1% DDM + 0.02% CHS, 12) 2% OG + 0.02% CHS. Where possible, detergents were diluted 10-fold in column buffers after solubilization, but were always kept at concentrations >2-fold above the critical micelle concentration (CMC) values. C) Coomassie–stained gels of HA-His8 tagged (HAR1H) and StrepII-His8 tagged (SR1H) CCR1 after two-column purification.
Figure 6
Figure 6. Representative size exclusion chromatography of CCR1 in different micelles
a) CCR1 was purified by affinity chromatography, concentrated and run on a size-exclusion column as described in procedures. The different oligomeric states are indicated. b) SDS-PAGE and western blot analysis is unable to distinguish between the presence of monomers, dimers and higher order species of CCR1 solubilized in DDM/CHS.
Figure 7
Figure 7. Binding and competition studies of CCR1 solubilized in micelles
a) Various concentrations of CCR1 solubilized in a number of different detergent/lipid micelles were incubated with a fixed concentration (5nM) of C-terminally fluorescein labeled chemokine (CCL7 or CCL14). Binding was monitored by increases in fluorescence anisotropy (Δr) values relative to those obtained in the absence of CCR1. b) 0.17μM CCR1 was incubated with increasing concentrations of the CCR1 inhibitor BX-471 for 15 minutes, before the addition of 5nM CCL14-fluorescein (n=3, shown as mean ± S.D.).
Figure 8
Figure 8. Stability of CCR1 over time in micelles
Samples of CCR1 in DDM/CHS micelles were placed at different temperatures for the indicated times, before mixing with CCL14-fluorescein and measuring binding by fluorescence anisotropy. Loss of function over time is indicated by decreasing values on the y-axis (n=3, shown as mean ± S.D.).
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